To Explore The Expression And Clinical Significance Of SNHG3 In Hepatocellular Carcinoma

Background &AimLong noncoding RNAs (lncRNAs) have been found dysregulated in human disease, especially in cancer. Cancer genomes are found disorganized and unstable, which embody numerous somatic copy-number alterations (SCNAs). Although genomic instability is the primary cause for the large portion of the copy number abnormalities, tumorigenesis can be attributable to a portion of SCNAs. Cancer-driving SCNA region have been successfully identified by systemic analyses using genome-wide functional screening and large-scale genomic profiles. However, many focal SCNAs in cancer have been mapped to regions characterized by protein-coding gene desert while the proportion of protein-coding sequences is merely less than 2% of the human genome.Recently, mounting evidence has also revealed that a substantial part of the whole human genome show transcriptional potential despite the fact that only a small portion is protein-coding genes. This observation has led a growing recognition that human genomes consist of numerous long noncoding RNA genes. Additionally, lncRNAs have exerted important roles in development and disease in series of different cell types, implicated as regulators of diverse cellular processes including regulation of cell cycle, RNA stability, and chromatin structure, suggesting a ubiquitous role in regulation of cellular fate. However, the molecular mechanisms by which most lncRNAs act remain mysterious. Thus, further efforts toward definite mechanisms and functional significance of lncRNAs will be critical for our understanding of these processes on human cancer.Small nucleolar RNA host gene 3 (SNHG3) is an lncRNA whose potential function and mechanism in hepatocellular carcinoma (HCC) remain largely unknown. SNHG3, also known as a host gene of U17 (U17HG), is located at 1p36.1. The host gene for intronic U17 small nucleolar RNAs was characterized previously by Pelczar P. Comparison of U17HG genes between human and mouse has revealed that snoRNA-encoding intron sequences other than exon sequences are conserved between the two species and that neither human nor mouse spliced U17HG poly(A)+ RNAs have protein-coding potential. SNHG3 was shown to be up-regulated in human HCCs from microarrays by comparing expression profiles of lncRNAs in HCC samples and adjacent nontumor tissues from 7 patients in our previous research. However, research on the potential roles of SNHG3 is limited and it remains unknown whether the lncRNA’s overexpression implies functional significance on HCC.In the present study, we aimed to determine SNHG3 expression and its clinical significance in HCC. We analyzed SNHG3 expression in 51 pairs of HCC (cohort 1) by qRT-PCR and performed ISH to measure its expression in another group of 144 paired paraffin-embedded HCC samples (cohort 2). We then confirmed the association between SNHG3 expression and clinicopathological features in 144 HCC patients (cohort 2). Finally, based on multivariate analysis using the Cox Proportional hazards model combined with association between the expression of SNHG3 and the duration of individual patients’ overall survival (OS), recurrence-free survival (RFS), and disease-free survival (DFS), we validated the prognostic value of SNHG3 on HCC patients.Materials and Methods1. Tissue Samples and Patients DataAll tissue samples were obtained from consenting patients in accordance with protocols passed through the ethics committee of the Nanfang Hospital, Nanfang Medical University (Guangzhou, China).195 HCC patients from two independent cohorts were enrolled in this study. In cohort 1,51 pairs of fresh HCC samples were collected from patients undergoing initial hepatectomy in our hospital between January 2011 and July 2012. Specimens were preserved in liquid nitrogen immediately and stored at-80 ℃ until RNA extraction. In cohort 2,144 pairs of formalin-fixed, paraffin-embedded HCC samples were collected from patients undergoing initial hepatectomy in our hospital between January 2006 and July 2009. None of these samples were collected after any anticancer treatments, including radiotherapy, chemotherapy and surgery. No bias against selection for the HCC samples was introduced in this study. All patients from cohort 2 were taken on five-year follow-up. The clinical information of all patients is shown in Supplementary Table S1 and Supplementary Table S2. Tumor differentiation was determined according to the criteria suggested by Edmonson and Steiner, and tumor stage was defined based on the Barcelona Clinic Liver Cancer (BCLC) staging system.2. RNA Isolation, Reverse Transcription, and qRT-PCRTotal RNA was isolated from tissue samples using TRIzol(?) reagent (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA with a reverse transcription (RT) reagent kit (Takara Biotechnology, Dalian, China). qRT-PCR was performed by the SYBR(?) Green PCR Kit produced from Takara Biotechnology (Takara, Dalian, China). Conditions for SNHG3 amplification were 30s at 95 ℃ of preliminary denaturation followed by 40 cycles of 95 ℃ for 5s,60 ℃ for 34s. Specificity and efficiency were verified for all qPCR primers. Relative quantification (2-ΔΔCt) method was used for calculating fold changes.3. In situ hybridizationIn situ detection for SNHG3 was performed using the ISH Kit (Boster Bio-Engineering Company, Wuhan, China) based on the manufacturer’s instructions. Slides were scored by 2 pathologists respectively through microscopy according to both the intensity and proportion of SNHG3-positive cells. The staining intensity was defined as follows as the score standard:0 (negative),1 (weak),2 (medium),3 (strong). The staining extent was scored as 0 (0%),1 (1%-25%),2 (26%-50%),3 (51%-75%), and 4 (76%-100%). The final score of SNHG3 expression was calculated with the intensity+extent score, ranging from 0 to 7. The high-expression group (positive group) was categorized with a total score of 3 or higher.4. TCGA databaseIn order to verify the expression of SNHG3 in HCC, we used RNA-seq analysis to identify the expression of SNHG3 in HCC cohorts from The Cancer Genome Atlas (TCGA, https://tcga-data.nci. Nih. gov/tcga/). In total,401 samples were available for SNHG3 expression data representing 50 normal liver tissues and 351 HCC tissues. We collected the SNHG3 expression data from each case to compare the SNHG3 expression level in HCC tissues versus normal liver tissues.5. Oncomine databaseA set of microarray data in Wurmbach Liver was downloaded from the Oncomine Compendium of Expression Array data (www.oncomine.org).45 liver tissue samples were analyzed on Affymetrix U133 Plus 2.0 microarrays, including 35 HCC tissues and 10 normal tissues. The SNHG3 expression level was compared between HCC tissues and normal liver tissues. The microarray data had been deposited on UCSC under the accession number AJ006835.6. Statistical analysisAll statistical analysis was achieved by SPSS software (Abbott Laboratories, Chicago, IL). Kaplan-Meier and the log-rank test were used to calculate the survival curves. The influence of variables on survival was evaluated by univariate and multivariate Cox proportional hazards model. Each variable with statistical significance on univaviate analysis was entered into multivariate models for estimating their independent prognostic value on cancer. The Mann-Whitney U-test was applied to assess the relationship between SNHG3 expression and clinicopathological characteristics. The analysis of qRT-PCR and datasets was performed by Student’s t-test. P< 0.05 was identified as statistical significance.Results1. Identification of SNHG3 transcripts expression in HCC cell lines and tissuesWe examined the two transcripts expression data from a panel of HCC cell lines by qRT-PCR, and we found that the 2238-bp transcript was more abundant in all six HCC cell lines, while the 2346-bp transcript was barely detected or lowly expressed (Fig.1a). To further confirm the result, we also detected the SNHG3 transcripts expression in 4 paired human HCC tissues and adjacent noncancerous tissues by qRT-PCR (Fig.1b), which was consistent with the data of cellular level.2. SNHG3 overexpression in HCC tissuesThe expression level of SNHG3 was about 5-fold higher in the tumor tissues compared with the adjacent noncancerous tissues from 51 HCC patients by qRT-PCR. Additionally, a microarray analysis from Wurmbach Liver using Oncomine database (accession AJ006835, Wurmbach,2007) and TCGA dataset indicated that SNHG3 was expressed more highly in HCC tissues than normal tissues. To further confirm these results, SNHG3 was expressed significantly higher in 91 (63.2%) cancer samples than controls determined by ISH in another group of 144 paired paraffin-embedded HCC samples (cohort 2).3. Correlation between SNHG3 expression and clinicopathological features in HCC patientsWe analyzed the association between SNHG3 expression and clinicopathological features in 144 HCC patients (cohort 2). SNHG3 expression was associated with tumor size (P=0.003), PVTT (P=0.014), and relapse (P=0.038). However, SNHG3 expression was not associated with gender, age, liver cirrhosis, BCLC stage.4. SNHG3 overexpression was an independent unfavorable prognostic factor in HCCThrough Kaplan-Meier and Log-rank analysis, SNHG3 expression was negatively correlated with OS and RFS and DFS rates in HCC patients. Furthermore, univariate analysis of prognostic variables revealed that SNHG3 expression (P< 0.001), portal vein tumor thrombus (P=0.004), relapse (P<0.001), BCLC stage (P=0.003), and tumor size (P=0.014) were significantly related to overall survival (Table 2). Multivariate analysis for all of the significant variables in the univariate using the Cox Proportional hazards model showed that SNHG3 expression was an independent prognostic factor for HCC patients (P<0.001, Table 2).Conclusions1. SNHG3 is significantly upregulated in human HCC tissues compared with adjacent noncancerous tissues.2. SNHG3 expression was positively associated with tumor size, portal vein tumor thrombus, and relapse.3. HCC patients with SNHG3 overexpression have worse prognosis than those with low expression of SNHG3.4. SNHG3 was an independent prognostic factor for HCC patients.